Male-derived copulatory plugs enhance implantation success in female Mus musculus†

Among a wide diversity of sexually reproducing species, male ejaculates coagulate to form what has been termed a copulatory plug. A number of functions have been attributed to copulatory plugs, including, but not limited to, the inhibition of female remating and the promotion of ejaculate movement. Here we demonstrate that copulatory plugs also influence the likelihood of implantation, which occurs roughly four days after copulation in mice. Using a bead transfer method to control for differences in ejaculate retention and fertilization rates, we show that implantation rates significantly drop among females mated to genetically engineered males incapable of forming plugs (because they lack functional TGM4, the main enzyme responsible for its formation). Surprisingly, this result does not correlate with differences in circulating progesterone levels among females, an important hormone involved in implantation. In this paper we discuss three models that connect male-derived copulatory plugs to implantation success, including the hypothesis that plugs contribute to a threshold amount of stimulation required for females to become receptive to implantation.

93 Oestrous Synchronisation Studies in a Marsupial

There is no strategy to precisely synchronise and predict oestrus and ovulation in marsupials. This is the major limitation in applying assisted reproduction to marsupial conservation. Routine methods of female synchronization that target the ovary (effective in eutherians) are ineffective in marsupials because they do not disrupt the corpus luteum. Gondaotropin-releasing hormone (GnRH) agonists have been used to suppress female cycling in several marsupials and work by targeting GnRH action in the pituitary. After the GnRH agonist reaches its active endpoint, female cycling returns. This study investigated whether Lucrin Depot (Abbott Laboratories, Lake Bluff, IL, USA), a tailored 1-month microsphere GnRH agonist preparation, has the potential to first suppress female cycling, and then facilitate a synchronous return to female cycling in a model marsupial, the tammar wallaby (Macropus eugenii). Preliminary studies indicated that Lucrin could be used to regulate ovarian activity, but a dose >0.20 mg kg−1 was required. In this study, nonpregnant tammars (n = 18, 6 per group) had their pouch young removed (RPY) on Day 0; RPY suspends lactation and reactivates cycling, allowing for controlled assessment of oestrus and mating in treated and untreated females. On Day 0, an intramuscular (IM) injection of water (Control) or 1 mg kg−1 of Lucrin Depot (group A) was given after RPY. Group B also received 1 mg kg−1 of Lucrin Depot but this was not injected until Day 10 after RPY (a strategy to assess whether timing of Lucrin yields different results). Females were placed in breeding pens of 1 male to 3 females. Reproductive suppression and synchrony of return to female cycling was assessed by pouch and urogenital opening checks to detect newborn young and copulatory plugs. All data (time of mating/conception) were analysed using ANOVA (significance, P < 0.05). There was a significant difference between the first mating response in the Control compared with Groups A and B (P = 0.0003), but the time from Lucrin injection to mating, Day 0 or Day 10, was not significant (P = 0.1431). All control females mated before Day 32 RPY, whereas all Lucrin-treated females mated from Day 33 to 66. All Lucrin-treated females conceived as evidenced by pouch young. Further analysis revealed 2 Lucrin-injection to mating response intervals, those females delayed by ~10 days that conceived between 33 and 50 days (n = 7: 41 ± 2.1 days; mean ± SEM), and those females delayed by ~30 days that conceived between 61 and 66 days (n = 5: 62 ± 1.0 days; mean ± SEM) following injection. Thus, an IM injection of 1 mg kg−1 of Lucrin Depot is sufficient to delay oestrus in breeding tammar wallabies whether given at the time of RPY or 10 days later. This study confirmed that Lucrin Depot can be used to regulate ovarian activity in macropod marsupials, and has potential to form the basis of a GnRH agonist-based synchronisation strategy for use in assisted breeding for marsupial conservation. This research was supported by Holsworth Wildlife Research Endowment.

Absorption and distribution of estradiol from male seminal emissions during mating

Estradiol-17β (E2) plays critical roles in female maturation, sexual receptivity, ovulation and fertility. In many mammals, contact with males can similarly affect these female parameters, whereas male excretions contain significant quantities of E2. We administered radiolabeled estradiol ([3H]E2) to male mice in doses representing a small fraction of their endogenous E2. These males were paired with sexually receptive females, and radioactivity was traced into the females’ systems. In Experiment 1, males were given [3H]E2 at 24 and 1 h before mating. Male-to-female [3H]E2 transfer intensified with increasing numbers of intromissions and spiked in the uterus after insemination. In Experiment 2, sexually experienced young males received [3H]E2 at 72 and 24 h before mating, and all mated to ejaculation. The copulatory plug deposited in the female reproductive tract contained substantial levels of radioactivity. The uteri, other tissues and blood serum of females displayed radioactivity indicative of E2 transfer. In Experiment 3, radioactivity was observed 3 and 18 h after insemination in the females’ uteri and other tissues, including parts of the brain. In Experiment 4, we observed substantial levels of radioactivity in semen as well as the copulatory plugs retrieved from the females after mating. Transferred E2 could directly affect abundant estrogen receptors in the female reproductive tract without potential metabolism by the liver. Sexually transferred E2 may facilitate uterine preparation for blastocyst implantation. These data converge with several lines of evidence indicating that male-sourced E2 can transfer to proximate females in bioactive form, contributing to various mammalian pheromonal effects.

Sexual conflict over mating in red-sided garter snakes ( Thamnophis sirtalis ) as indicated by experimental manipulation of genitalia

Sexual conflict over mating can result in sex-specific morphologies and behaviours that allow each sex to exert control over the outcome of reproduction. Genital traits, in particular, are often directly involved in conflict interactions. Via genital manipulation, we experimentally investigated whether genital traits in red-sided garter snakes influence copulation duration and formation of a copulatory plug. The hemipenes of male red-sided garter snakes have a large basal spine that inserts into the female cloaca during mating. We ablated the spine and found that males were still capable of copulation but copulation duration was much shorter and copulatory plugs were smaller than those produced by intact males. We also anaesthetized the female cloacal region and found that anaesthetized females copulated longer than control females, suggesting that female cloacal and vaginal contractions play a role in controlling copulation duration. Both results, combined with known aspects of the breeding biology of red-sided garter snakes, strongly support the idea that sexual conflict is involved in mating interactions in this species. Our results demonstrate the complex interactions among male and female traits generated by coevolutionary processes in a wild population. Such complexity highlights the importance of simultaneous examination of male and female traits.